Our overall goal is to study red blood cell production, terminal red cell differentiation and erythroid specific gene expression through genetic studies. Our basic approaches are to study disease causing mutations or to use transgenic mouse technology to create null animals by gene replacement strategies (knock out) or to introduce specific mutations into red cell genes by knock in strategies. Our work is divided into 3 specific aims. Specific Aim 1: We will use SNP-CGH and whole-exome sequencing to discover novel mutations associated with Diamond Blackfan Anemia. We have initiated a collaboration with the Diamond Blackfan Anemia registry to receive DNA samples from patients whose mutation has not been detected by sequencing of known DBA genes. We predict that we will discover both copy number variations and atypical mutations that can help explain the failure of erythropoiesis in these patients. We are also desigining reporter plasmids to detect stress induced changes in ribosomal protein mRNA translation. These reporters will be used for a small molecule screen to identify drugs that can be used to treat DBA. Specific Aim 2: To generate a comprehensive profile of chromatin changes associated with the activation of specific ankyrin promoters. Regulatory elements such as promoters, barriers and enhancers often co-localize with DNsae I hypersensitive sites (HSs). We have developed a high throughput quantitative PCR assay to detect DNase I HS across a 119 kb region of the Ank-1 promoter region, which includes a neuromuscular, an erythroid and a ubiquitous promoter. We have identified 6 discrete HSs within the region, which flank the three promoters. We have shown that those surrounding the erythroid promoter are barrier elements, but do not have either enhancer or enhancer blocking activity. We will use the chromatin conformation capture (carbon copy) assay (5C) to determine how these HSs interact with each other. We have already shown that the sites surrounding the erythroid promoter are in contact in erythroid cells, but separated in non-erythroid cells. We will identify the proteins associated with the HSs by chromatin immune precipitation (ChIP). We hypothesize that the deletion of the erythroid promoter may allow one of the other promoters to become active in erythroid cells. To test this we have developed a targeted deletion of this region in ES cells for evaluation. Specific Aim 3: Structure/Function analysis of band 3 protein in red cells. Band 3 (B3) is an integral membrane protein that serves as the major anion channel for red blood cells and binds ankyrin, tethering the actin/spectrin network to the red cell membrane. In addition to binding ankyrin, Band 3 is the major anion exchange protein of the red cell membrane and plays a critical role in maintaining red cell hydration, which is important to prevent the concentration dependent polymerization of deoxy HbS. We have shown that the N-terminus of band 3 lies in the cytoplasm and is a high affinity binding site for deoxy hemoglobin, and that this binding serves as a catalyst for HbS polymerization. Because of this we are preparing to knock out this Hb binding region to determine the physiological consequences of disrupting this association. Analysis of red cell membranes will be performed to determine whether this association alters red cell stability. We will breed these animals to mouse models of SCD to determine whether preventing deoxy Hb binding to band 3 can modulate the severity of SCD.